Identification of the Binding Epitope of an Anti-Mouse CCR6 Monoclonal Antibody (C6Mab-13) Using 1× Alanine Scanning

CC chemokine receptor 6 (CCR6) is one of the members of the G-protein-coupled receptor (GPCR) family that is upregulated in many immune-related cells, such as B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. The coordination between CCR6 and its ligand CC motif chemokine ligand 20 (CCL20) is deeply involved in the pathogenesis of various diseases, such as cancer, psoriasis, and autoimmune diseases. Thus, CCR6 is an attractive target for therapy and is being investigated as a diagnostic marker for various diseases. In a previous study, we developed an anti-mouse CCR6 (mCCR6) monoclonal antibody (mAb), C6Mab-13 (rat IgG1, kappa), that was applicable for flow cytometry by immunizing a rat with the N-terminal peptide of mCCR6. In this study, we investigated the binding epitope of C6Mab-13 using an enzyme-linked immunosorbent assay (ELISA) and the surface plasmon resonance (SPR) method, which were conducted with respect to the synthesized point-mutated-peptides within the 1–20 amino acid region of mCCR6. In the ELISA results, C6Mab-13 lost its ability to react to the alanine-substituted peptide of mCCR6 at Asp11, thereby identifying Asp11 as the epitope of C6Mab-13. In our SPR analysis, the dissociation constants (KD) could not be calculated for the G9A and D11A mutants due to the lack of binding. The SPR analysis demonstrated that the C6Mab-13 epitope comprises Gly9 and Asp11. Taken together, the key binding epitope of C6Mab-13 was determined to be located around Asp11 on mCCR6. Based on the epitope information, C6Mab-13 could be useful for further functional analysis of mCCR6 in future studies.

In this study, we performed an epitope identification of a rat anti-mCCR6 mAb (C 6 Mab-13; IgG 1 , kappa) using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) analysis against the alanine-substituted N-terminal peptides of mCCR6.

Peptides
Sequences

Measurement of Dissociation Constants Using Surface Plasmon Resonance (SPR)
The dissociation constants (K D ) between C 6 Mab-13 and the epitope region peptides were measured using SPR. C 6 Mab-13 was immobilized on the CM5 sensor chip according to the manufacturer's protocol (Cytiva, Marlborough, MA, USA). In summary, C 6 Mab-13 was diluted to 10 µg/mL by an acetate buffer (pH 4.0; Cytiva) and immobilized using an aminecoupling reaction. The surface of flow cell 2 of the CM5 sensor chip was treated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide (NHS), followed by an injection of C 6 Mab-13. The unreacted NHS-ester was blocked with ethanolamine after C 6 Mab-13 immobilization. The K D between C 6 Mab-13 and mCCR6 peptides (50, 25, 12.5, 6.25, and 3.13 µM) were measured using Biacore X100 (Cytiva) at 25 • C. The buffer was filtrated with PBS containing 0.05% (v/v) Tween 20 and 0.24% (v/v) dimethyl sulfoxide (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The single-cycle kinetics method was used to measure the binding signals. The data were analyzed using 1:1 binding kinetics to determine the association rate constant (k a ), dissociation rate constant (k d ), and K D using BIAevaluation software (Cytiva).

Epitope Identification of C 6 Mab-13 by SPR Using 1× Alanine-Substituted mCCR6 Peptides
To confirm the C 6 Mab-13 epitope, we measured the binding affinity between C 6 Mab-13 and the synthesized peptides, including 20 point mutants and the WT of mCCR6, using Biacore Antibodies 2023, 12, 32 5 of 9 X100. The peptides' sequences are presented in Table 1, and the measured values are summarized in Table 2. The k a , k d , and K D of G9A and D11A were not determined. These results demonstrated that Gly9 and Asp11 were the critical amino acids of the C 6 Mab-13 epitope.

Discussion
This study examined the binding epitope of C 6 Mab-13 through a 1× alanine-substitutedpeptide-scanning method using ELISA and SPR. We concluded that Asp11 is a pivotal epitope aa in ELISA, while Gly9 and Asp11 are critical in SPR. This epitope is located outside the region of all three extracellular domains of CCR6 and N-terminal residues from Tyr27 to Leu38, to which the chemokine ligand CCL20 binds [59,60]. There is a possibility that structural changes might occur upon C 6 Mab-13 s binding to CCR6, which leads to allosteric effects on CCL20 binding. Therefore, we will investigate the neutralizing activity of C 6 Mab-13 between CCL20 and CCR6 in the future study.
A recent report showed that low rather than high affinity of mAb to a target provokes elevated activity through inducing the clustering of receptors. These findings provide new insights for antibody-mediated receptor signaling [61]. Since CCR6 is involved in intracellular signaling [62], the relationship between antibody affinity and the effect of cellular signaling should be investigated in future studies.
The epitope-mapping results obtained using ELISA ( Figure 1) and SPR (Table 2) indicated a similar region of mCCR6 as the binding epitope. However, Gly9 was only identified as the critical aa by via SPR analysis ( Table 2). The experimental system differs between both experiments, as follows: (i) for ELISA, the synthesized peptides were immobilized on immunoplates, while C 6 Mab-13 was immobilized on a CM5 sensor chip in the SPR analysis; (ii) the reaction times between the antigen and the antibody were different; and (iii) the secondary antibody was only used for ELISA. These different conditions may have precipitated the inconsistent results of both experiments in this study.
In the SPR analysis, mutant peptides of F8A, T10A, Y13A, and D14A increased the K D values by 15.5-, 4.4-, 16.5-, and 2.8-fold, respectively (Table 2). These results indicate that Phe8, Thr10, Tyr13, and Asp14 may contribute to C 6 Mab-13 s binding to mCCR6. In the future, we will adopt the cell-based alanine-or 2× alanine-scanning methods for a detailed epitope analysis of C 6 Mab-13, as we have clarified the epitope of mAb [63].
When CCL20 is secreted in tumor tissues [64], it attracts CCR6-expressing Treg cells [65], which are involved in tumor progression and poor prognosis [66,67]. Therefore, novel cancer treatment strategies using CCR6-expressing chimeric antigen receptor-T (CAR-T) cells have been designed [68,69]. Furthermore, removing immunosuppressive cells, such as CCR6+ Treg cells, may enhance antitumor efficacy [70]. In this study, we demonstrated that C 6 Mab-13 possesses high binding affinity against mCCR6, which was expressed in Chinese hamster ovary-K1 cells (K D : 2.8 × 10 −9 M according to flow cytometric analysis) [52]. Therefore, C 6 Mab-13 is expected to be useful for antitumor evaluations considering the depletion of CCR6-expressing Treg cells in mouse models.

Institutional Review Board Statement:
Not applicable for studies not involving humans or animals.

Informed Consent Statement: Not applicable.
Data Availability Statement: All related data and methods are presented in this paper. Additional inquiries should be addressed to the corresponding authors.

Conflicts of Interest:
The authors declare no conflict of interest.